SERRA-BADRICH - HUMAN MICROBIOME (ROSACEA, ATOPIC DERMATITIS

Microbiome

Rosacea Atopic Dermati tis

Dra Esther Serra-Baldrich Dermatology Department ImmuneCutaneous Unit

A few facts : Human body: 10 13 cells Microbiota: 10 14 cells, 8x10 6 genes (vs 20.000 !) Weight: 1,5 kg (!)

CUTANEOUS MICROBIOTA

Whole of saprophytic and pathogenic microorganisms that colonize our body

Transitory flora

Resident flora stratum corneum

streptococcus neisseria pseudomona E coli C albicans S aureus

proteobacterias firmicutes bacteriadetes actinobacterias

Form microbial imprint 25% in hair follicles and sweat glands

Takes advantage of habitat modification to proliferate

An average healthy human body possesses 10 14 microorganisms – 10-fold the number of human cells.

These bacterial, viral, fungal and eukaryotic microorganisms are absolutely essential to everyday life

The gut microbiome is by far the most extensively studied. The human gut contains nearly 10 trillion bacterial cells and over 2000 different species The bacterial communities in other parts of the body are quite distinct from those in the gut, and differ significantly depending on body site differences and are influenced by pH, temperature and oxygen content.

MICROBIOTA

• A single square centimetre of the human skin can contain up to one billion microorganisms. A single square centimetre of the human skin can contain up to one billion microorganisms • Distinct skin microbial communities arise, becoming increasingly diverse over time. • These microbial niches and their inhabitants continue to transform over time with puberty, aging and environmental exposure Leyden JJ, McGinley KJ, Mills OH et al . Age-related changes in the resident bacterial flora of the human face. J. Invest. Dermatol. 1975; 65 : 379–81 .

• .

2008 The human microbiome project Has helped to characterise the skin microbiome

Studied healthy volunteers and discern how it varies across different niches, individuals and time

Small variations from one person to another The microbioma of the same places of different people is more similar than the one of another place

Our bacterial signature is

common at the family level but specific at the level of genera and species

• There is a specific relationship between the immune system of each tissue and the naturally colonizing bacteria

• There are skin diseases where there is a modification of the local microbiota – ACNE / ROSACEA (sebaceous area) – PSORIASIS (dry area) – ATOPIC dermatitis (wet area) There is a break in balance between inflammatory versus anti-inflammatory flora Resident bacteria are not passive to protect against infection but are a necessary element for the development of immunity

ROSACEA

• Rosacea is a chronic disease, with

evolution in outbreaks, characterized

by an inflammatory process and

vasculopathy.

• usually rosacea is primarily characterized by erythema of sev- eral and persistent degree, which worsens suddenly ( flushing), with or without papular inflammatory or even pustular lesions, similar to acne, and, in more chronic forms, of telangiectasias.

vascular reaction

GI alterations

environment

rosácea

food , spices, alcohol exposition

endotelium damage

barrier disruption

microorganisms

• National Rosacea Society (NRS) developed a classification according to the clinical findings, regardless of evolutionary or pathogenic aspects (erythematotelangiectatic, papulo- pustular, phymatous and ocular) and only one variant, the granulomatous .

Jansen T, Plewig G. Rosacea: classification and treatment. J R Soc Med. 1997;90:144-50.

• Rosacea may occur in patients with dry skin or skin with important seborrheic levels, not being necessarily linked to a skin type. • A recent study evaluated 135 patients with rosacea and without treatment, and showed that the erythematotelangiectatic form of the disease is more common in dry skin than in seborrheic skin.

Tan J, Blume-Peytavi U, Ortonne JP, Wilhelm K, Marticou L, Baltas E, et al. An observational cross-sectional survey of rosacea: clinical associations and progression between subtypes. Br J Dermatol. 2013;169:555-62.

• Papulopustular form

• sebaceous levels were normal, but with a significant reduction in hydration levels. • imbalance in the fatty acid concentration in sebum – myristic acid was present in higher concentration in sebum of these patients compared with a control group without the dermatosis – saturated long-chain fatty acids (behenic, tricosanoic and lignoceric), as well as monounsaturated cis-11-eicosanoic, were in lower concentrations

Ní Raghallaigh S, Bender K, Lacey N, Brennan L, Powell FC. The fatty acid profile of the skin surface lipid layer in papulopustular rosacea. Br J Dermatol. 2012;166:279-87

• these patients exhibit a modified innate immune response to environmental stimuli, favoring inflammation. Over time, these inflammatory outbreaks lead to dermal fibrotic conditions

• However, not all patients with erythematotelangiectatic form necessarily progress to phymatous or papulopustular form.

• The innate response involves the secretion of antimicrobial peptides (AMP), from the keratinocytes, sebocytes and mast cells. Expression of these peptides is strictly regulated, because although it is an important line of defense, it can also cause tissue damage.

• Among AMPs, the group of cathelicidins has received special attention in the physiopathology of rosacea.

• Cathelicidin LL-37 is an important molecule in the innate metabolism, and in ammatory diseases such as rosacea, there is a disturbance in its processing, which results in peptide fragments that cause inflammation

An Bras Dermatol. 2016;91(1):59-63.

Epidemiology 5% USA (16 million) Germany 2.2%

Sweden 10% Estonia 22% Prevalence increase from south to north Phototypes I and II Older adults . Women early onset. Fimas in menPoor QoL stigmatization, loss of self-esteem and anxiety

Berg M, Lidén S: An epidemiological study of rosacea. Acta Derm Venereol 1989, 69:419-23. Abram K, Silm H, Oona M: Prevalence of rosacea in an Estonian working population using a standard classification. Acta Derm Venereol 2010, 90:269-73. Böhm D, Schwanitz P, Stock Gissendanner S, Schmid-Ott G, Schulz W: Symptom severity and psychological sequelae in rosacea: Results of a survey. Psychol Health Med 2013; in press.

• Innate immunity altered • Neurogenic inflammation • Vascular changes • Environmental factors • Genetics: 30-40% patients have a relative with rosacea Pathophysiology

• High association of rosacea with sensitive skin • Concept of alteration of the barrier function in this entity • Restricted to facial area /// a DA • Increased TEWL activates epidermal proteases (serine proteases stratum corneum)

Innate immunity– microbiome expression PRR modified • Are innate immune receptors expressed by keratinocytes, which release cytokines, chemokines and AMP

PRRs • TLR Toll like receptors----TLR2…..IL-10 • NOD nucleotide oligomerization domain • NLR nucleotide like receptors • CLR c-type lectin receptors • GRR G-protein coupled receptors • RIG retinoid acid inducible gene • RLR retinoid I like receptors

• TLR2 Recognize Bacterial Wall Constituents

PRR = pattern recognition receptors PAMP = pathogen associated molecular patterns

In rosacea there is increased expression TLR 2 Hence greater susceptibility to stimulate innate immune system LL37 (AMP) increased levels in rosacea (it is angiogenic, antimicrobial and proinflammatory)

TLR2 +++

Increased activity KLK5

Keratinocyte +++Serin proteases production

Meyer-Hoffert U, Schröder J: Epidermal proteases in the pathogenesis of rosacea. J Investig Dermatol Symp Proc 2011, 15:16-23. Larrick JW, Hirata M, Zhong J, Wright SC: Anti-microbial activity of human CAP18 peptides. Immunotechnology 1995, 1:65-72.

LIVING AGENTS

Demodex Prevalence 60% approx.

It would be an aggravating factor, not causal

Hypersensitivity to Demodex Folliculorum

Distribution in the sebaceous follicle

Demodex folliculorum • Upper density 5 mites / cm2 • Can activate NOD-like receptors in vitro • NALP3 activates the 1ß inflamasome activating the immune response Bacillus oleronius • Endoespora in the mobile gram-negative • Its pathogenic potential unknown • Their proteins trigger proliferative response in mononuclear cells in peripheral blood

Staphylococcus epidermidis • Most prevalent commons of healthy skin • Presence associated with skin protection against pathogenic species • Potential pathogen only in wounds and medical devices inserted • Abundant presence in pustular lesions • Altered secretion profile Helicobacter pylori

• Controversial literature on its role • CagA (virulence factor) inconclusive

Chlamydophila pneumoniae

• Intracellular pathogen • Study single uncontrolled detection Ag C pneumoniae in 40% samples malarial biopsies and Ac in 80% of sera

Fdez-Obregon A, Patton Dl. The role of Chlamydia pneumoniae in the etiology of acne-rosacea: response to the use of oral azithromycin. Cutis 2007; 79: 163-7

Neurogenic inflammation Vascular changes

• Vascular endoltelial growth factor (VEGF) is present is the epidermis and is expressed by infiltrating cells • VEGF receptor-ligand binding contributes to vascular changes and cellular infiltrate • The intestinal vasoactive peptide (VIP) is increased in the serum and in the cutaneous blood vessels of the phymomatous R • LL37 induces cell-endothelial changes promoting vascular effects on rosacea

• Sust P has also been linked to rosacea

• Regulates local blood flow

• Induces mast cell degranulation

• Increased proinflammatory cytokines and chemokines CXCL8, TNF-α, IL-3

Journal of Investigative Dermatology Symposium Proceedings 15 , 2–11

• The affected skin of rosacea has a lower heat sensitivity threshold than normal skin. • Possible participation of transient potential receptors of vanilloide ions TRPV1 and ankirin 1 (TRPA1) ions as heat and cold cellular sensors • Its activation increases the release of sust P • The expression of TRPV1 genes is increased in type 1 rosacea • The immunoreactivity of TRPV2, TRPV4 and TRPV2 gene expression is increased in type 2 of rosacea • TRPV3 and TRPV4 increased in type 3

Environmental factors • Food: food temperature, alcohol • Stress as a trigger for seborrhea • Climate: temperature variations.

• Solar exposure as a triggering factorUV induces ROS formation (reactive oxygen species)TLR2-ROS interaction increases levels CXCL8 • Myeloid differentiation factor (MyD88) overexpressed

Atopic dermatitis

• Atopic dermatitis (AD) is characterized by intense itch, disruption of the skin barrier, and upregulation of type 2 (including type 2 helper T cell [Th2]) immune responses 1,2 • AD significantly impairs quality of life in both adults and children 3–5 • There is relatively little qualitative information on patient perception of AD and impact of the disease on their life

1. Brandt EB, Sivaprasad U. J Clin Cell Immunol. 2011:2 :pii 110. 2. Gittler JK, et al. J Allergy Clin Immunol. 2012;130:1344-54; 3. Sánchez-Pérez J, et al. Actas Dermosifiliogr. 2013;104:44-52. 4. Silverberg JI, et al. J Invest Derm. 2015;135:56-66. 5. Simpson EL, et al. J Am Acad Dermatol. 2016a;74:491-8.

• The most annoying symptom is a recalcitrant pruritus • There is an 'itch-scratch cycle’ response • Epidermal epithelial terminal differentiation anomalies • Contains a defective stratum corneum • Increased penetration of allergens and IgE sensitization

• Usual onset at early age apr.20% • Prevalence in adults 2-5% • Chronic and relapsing course • Requires safe and long-term treatment strategies

Cutaneous barrier

Clinical phenotype AD

Childhood onset, disappearance adolescence Childhood onset, persistent severe eczema Adolescent onset / adult mild eczema Adolescent onset / adult, severe eczema Intrinsic (IgE N) 20% Extrinsic (elevated IgE), Food / sensitization, aeroallergens Infection / colonization S aureus Disseminated infection (viral)

• Biomarkers to assess polarized immune end-points in patients with AD Defines patients who are TH2 polarized vs. non-TH2 IgE

Eosinophilia TSLP serum FUTURE:

epidermal proteomics Genomic Transcriptosomes gene Blood biomarkers

Microbiota –Levels influence DA

microbiota intestinal

microbiota cutaneous

Cutaneous Inflammatory manifestations

Early development immunity

Allergic manifestations

S aureus are present in 90% of patients with AD in healthy or affected skin Partly because of AMP deficit

The IL4 and. IL13 inhibit

expressionHBD-2 gene and LL-37

S aureus toxins act as super Ags triggering or aggravating the immune and inflammatory response

Also would be factors of resistance to corticosteroids

In addition, α-toxin induces keratinocyte death

The Role of the Skin Microbiome in Atopic Dermatitis: A Systematic Review R. Dybboe 1,* , J. Bandier 2 , L. Skov 2 , L. Engstrand 3 andJ. D. Johansen 1

• A systematic literature search was conducted October 21 2016 in PubMed, Embase, Scopus and ClinicalTrials.gov using search terms from the categories: Skin, microbiome and AD – without language and date limitations • In this systematic review demonstrated that AD skin in humans is characterised by low bacterial diversity and high non-Malassezia fungal diversity. On involved skin the bacterial diversity was even lower.

• The data is not sufficiently robust for good characterisation; however, dysbiosis in atopic dermatitis does not only implicate Staphylococcus species, but also microbes such as Propionibacterium and Malassezia. • A causal role of dysbiosis in eczema in mice encourages future studies to investigate if this applies in humans too.

• Other important aspects are temporal dynamics and the influence of methodology on microbiome data.

microbiome sampling at 3 points in the first 6 months of life at 4 skin sites relevant to AD: the antecubital and popliteal fossae, nasal tip, and cheek. They identified 10 infants with AD and compared them with 10 randomly selected control infants with no AD. They performed bacterial 16S ribosomal RNA sequencing and analysis directly from clinical sample

An early interaction between the microbiome and immune system via multiple routes (skin–gut–lung) could feasibly affect the risk of a subsequent development of atopic diseases. We understand that the composition of the fungal community may be important in AD, yet we do not know how this pattern changes in flares. Understanding the complex interactions of the human microbiome will aid the development of novel and more targeted treatments to modulate the microbiome and influence AD outcomes. Principles gleaned from the success of faecal transplantation in the treatment of C. difficile infections may be translated to AD with organism transplantation used to normalise the skin or gut of those with, or at high risk of developing AD.

Correcting dysbiosis to a symbiotic state is very important It can be achieved with non-pathogenic microorganisms

Lysat of Vitreoscilla filiformis + TLR2 with production of β defensins

Individualized treatment

Novak and Simon Allergy 2011; 66: 830–839